Long-term culture, genetic manipulation and xenotransplantation of human normal and breast cancer organoids

TY - JOUR

T1 - Long-term culture, genetic manipulation and xenotransplantation of human normal and breast cancer organoids

AU - Dekkers, Johanna F

AU - van Vliet, Esmée J

AU - Sachs, Norman

AU - Rosenbluth, Jennifer M

AU - Kopper, Oded

AU - Rebel, Heggert G

AU - Wehrens, Ellen J

AU - Piani, Carol

AU - Visvader, Jane E

AU - Verissimo, Carla S

AU - Boj, Sylvia F

AU - Brugge, Joan S

AU - Clevers, Hans

AU - Rios, Anne C

PY - 2021/4

Y1 - 2021/4

N2 - Organoid technology has revolutionized the study of human organ development, disease and therapy response tailored to the individual. Although detailed protocols are available for the generation and long-term propagation of human organoids from various organs, such methods are lacking for breast tissue. Here we provide an optimized, highly versatile protocol for long-term culture of organoids derived from either normal human breast tissues or breast cancer (BC) tissues, as well as culturing conditions for a panel of 45 biobanked samples, including BC organoids covering all major disease subtypes (triple-negative, estrogen receptor-positive/progesterone receptor-positive and human epidermal growth receptor 2-positive). Additionally, we provide methods for genetic manipulation by Lipofectamine 2000, electroporation or lentivirus and subsequent organoid selection and clonal culture. Finally, we introduce an optimized method for orthotopic organoid transplantation in mice, which includes injection of organoids and estrogen pellets without the need for surgery. Organoid derivation from tissue fragments until the first split takes 7-21 d; generation of genetically manipulated clonal organoid cultures takes 14-21 d; and organoid expansion for xenotransplantation takes >4 weeks.

AB - Organoid technology has revolutionized the study of human organ development, disease and therapy response tailored to the individual. Although detailed protocols are available for the generation and long-term propagation of human organoids from various organs, such methods are lacking for breast tissue. Here we provide an optimized, highly versatile protocol for long-term culture of organoids derived from either normal human breast tissues or breast cancer (BC) tissues, as well as culturing conditions for a panel of 45 biobanked samples, including BC organoids covering all major disease subtypes (triple-negative, estrogen receptor-positive/progesterone receptor-positive and human epidermal growth receptor 2-positive). Additionally, we provide methods for genetic manipulation by Lipofectamine 2000, electroporation or lentivirus and subsequent organoid selection and clonal culture. Finally, we introduce an optimized method for orthotopic organoid transplantation in mice, which includes injection of organoids and estrogen pellets without the need for surgery. Organoid derivation from tissue fragments until the first split takes 7-21 d; generation of genetically manipulated clonal organoid cultures takes 14-21 d; and organoid expansion for xenotransplantation takes >4 weeks.

KW - Animals

KW - Biological Specimen Banks

KW - Breast/pathology

KW - Breast Neoplasms/pathology

KW - Cell Culture Techniques/methods

KW - Clone Cells

KW - Female

KW - Genetic Techniques

KW - Humans

KW - Mice

KW - Organoids/pathology

KW - Time Factors

KW - Transplantation, Heterologous

U2 - 10.1038/s41596-020-00474-1

DO - 10.1038/s41596-020-00474-1

M3 - Article

C2 - 33692550

SN - 1754-2189

VL - 16

SP - 1936

EP - 1965

JO - Nature Protocols

JF - Nature Protocols

IS - 4

ER -